Calcilytic compounds

ABSTRACT

Novel calcilytic compounds and methods of using them are provided.

FIELD OF INVENTION

The present invention relates to novel calcilytic compounds,pharmaceutical compositions containing these compounds and their use ascalcium receptor antagonists.

In mammals, extracellular Ca²⁺ is under rigid homeostatic control andregulates various processes such as blood clotting, nerve and muscleexcitability, and proper bone formation. Extracellular Ca²⁺ inhibits thesecretion of parathyroid hormone (“PTH”) from parathyroid cells,inhibits bone resorption by osteoclasts, and stimulates secretion ofcalcitonin from C-cells. Calcium receptor proteins enable certainspecialized cells to respond to changes in extracellular Ca²⁺concentration.

PTH is the principal endocrine factor regulating Ca²⁺ homeostasis in theblood and extracellular fluids. PTH, by acting on bone and kidney cells,increases the level of Ca²⁺ in the blood. This increase in extracellularCa²⁺ then acts as a negative feedback signal, depressing PTH secretion.The reciprocal relationship between extracellular Ca²⁺ and PTH secretionforms an important mechanism maintaining bodily Ca²⁺ homeostasis.

Extracellular Ca²⁺ acts directly on parathyroid cells to regulate PTHsecretion. The existence of a parathyroid cell surface protein whichdetects changes in extracellular Ca²⁺ has been confirmed. See Brown etal., Nature 366:574, 1993. In parathyroid cells, this protein, thecalcium receptor, acts as a receptor for extracellular Ca²⁺, detectschanges in the ion concentration of extracellular Ca²⁺, and initiates afunctional cellular response, PTH secretion.

Extracellular Ca²⁺ influences various cell functions, reviewed in Nemethet al., Cell Calcium 11:319, 1990. For example, extracellular Ca²⁺ playsa role in parafollicular (C-cells) and parathyroid cells. See Nemeth,Cell Calcium 11:323, 1990. The role of extracellular Ca²⁺ on boneosteoclasts has also been studied. See Zaidi, Bioscience Reports 10:493,1990.

Various compounds are known to mimic the effects of extra-cellular Ca²⁺on a calcium receptor molecule. Calcilytics are compounds able toinhibit calcium receptor activity, thereby causing a decrease in one ormore calcium receptor activities evoked by extracellular Ca²⁺.Calcilytics are useful as lead molecules in the discovery, development,design, modification and/or construction of useful calcium modulatorswhich are active at Ca²⁺ receptors. Such calcilytics are useful in thetreatment of various disease states characterized by abnormal levels ofone or more components, e.g., polypeptides such as hormones, enzymes orgrowth factors, the expression and/or secretion of which is regulated oraffected by activity at one or more Ca²⁺ receptors. Target diseases ordisorders for calcilytic compounds include diseases involving abnormalbone and mineral homeostasis.

Abnormal calcium homeostasis is characterized by one or more of thefollowing activities: an abnormal increase or decrease in serum calcium;an abnormal increase or decrease in urinary excretion of calcium; anabnormal increase or decrease in bone calcium levels (for example, asassessed by bone mineral density measurements); an abnormal absorptionof dietary calcium; an abnormal increase or decrease in the productionand/or release of messengers which affect serum calcium levels such asPTH and calcitonin; and an abnormal change in the response elicited bymessengers which affect serum calcium levels.

Thus, calcium receptor antagonists offer a unique approach towards thepharmacotherapy of diseases associated with abnormal bone or mineralhomeostasis, such as hypoparathyroidism, osteosarcoma, periodontaldisease, fracture healing, osteoarthritis, rheumatoid arthritis, Paget'sdisease, humoral hypercalcemia associated with malignancy and fracturehealing, and osteoporosis.

SUMMARY OF THE INVENTION

The present invention comprises novel calcium receptor antagonistsrepresented by Formula (I) hereinbelow and their use as calcium receptorantagonists in the treatment of a variety of diseases associated withabnormal bone or mineral homeostasis, including but not limited tohypoparathyroidism, osteosarcoma, periodontal disease, fracture healing,osteoarthritis, rheumatoid arthritis, Paget's disease, humoralhypercalcemia associated with malignancy and fracture healing, andosteoporosis.

The present invention further provides a method for antagonizing calciumreceptors in an animal, including humans, which comprises administeringto an animal in need thereof an effective amount of a compound ofFormula (I), indicated hereinbelow.

The present invention further provides a method for increasing serumparathyroid levels in an animal, including humans, which comprisesadministering to an animal in need thereof an effective amount of acompound of Formula (I), indicated herein below.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention are selected from Formula (I)herein below:

wherein:

-   A represents C or N with one or two N in ring I;-   D represents C or N with one or two N in the ring II that is    attached at position 4 or 5 to ring I as indicated;-   X is selected from the group consisting of CN, NO₂, Cl, F, and H;-   Y is selected when A is C from the group consisting of Cl, F, Br, I    and H;-   Q is selected when D is C from the group consisting of H, R₁,    SO₂R₁′,-   R₁C(O)OR′₁, tetrazole, CH₂OH, COH, SO₂NR₁′R₁″, C(O)N R₁′R₁″, and-   NR₁SO₂R₁′, wherein R₁ is independently selected from the group    consisting of bond, hydrogen, C₁₋₄ alkyl, and optionally substituted    alkyl;-   R₁′, and R₁″ are independently selected from the group consisting of    hydrogen, C₁₋₄ alkyl, and optionally substituted alkyl, or R₁′, and    R₁″ together form a 3 to 7 membered optionally substituted    heterocyclic ring; and

Ar is phenyl or naphthyl, unsubstituted or substituted, heteroaryl orfused heteroaryl, such that the hetero-ring may contain N, O or S andmay be aromatic, dihydro or tetrahydro, unsubstituted or substituted.

As used herein, “alkyl” refers to an optionally substituted hydrocarbongroup joined by single carbon-carbon bonds and having 1-20 carbon atomsjoined together. The alkyl hydrocarbon group may be linear, branched orcyclic, saturated or unsaturated. Preferably, substituents on optionallysubstituted alkyl are selected from the group consisting of aryl, CO₂R,CO₂NHR, OH, OR, CO, NH₂, halo, CF₃, OCF₃ and NO₂, wherein R representsH, C₁₋₄ alkyl C₃₋₆ cycloalkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl,heterocycloalkyl, aryl or aryl C₁₋₄ alkyl; Additional substituents areselected from F, Cl, Br, I, N, S and O. Preferably, no more than threesubstituents are present. More preferably, the alkyl has 1-12 carbonatoms and is unsubstituted. Preferably, the alkyl group is linear.

As used herein “cycloalkyl” refers to optionally substituted 3-7membered carbocyclic rings wherein any substituents are selected fromthe group consisting of, F, Cl, Br, I, N(R₄)₂, SR₄ and OR₄, unlessotherwise indicated.

As used herein, “aryl” refers to an optionally substituted aromaticgroup with at least one ring having a conjugated pi-electron system,containing up to two conjugated or fused ring systems. Aryl includescarbocyclic aryl, and biaryl groups, all of which may be optionallysubstituted. Preferred aryl include phenyl and naphthyl. More preferredaryl include phenyl. Preferred substituents are selected from the groupconsisting of halogen, C₁₋₄ alkyl OCF₃, CF₃, OMe, CN, OSO₂ R and NO₂,wherein R represents C₁₋₄ alkyl or C₃₋₆ cycloalkyl.

As used herein, “alkenyl” refers to an optionally substitutedhydrocarbon group containing at least one carbon-carbon double bond andcontaining up to 5 carbon atoms joined together. The alkenyl hydrocarbonchain may be straight, branched or cyclic. Any substituents are selectedfrom the group consisting of halogen, C₁₋₄ alkyl, OCF₃, CF₃, OMe, CN,OSO₂ R and NO₂, wherein R represents C₁₋₄ alkyl or C₃₋₆ cycloalkyl.

As used herein, “alkynyl” refers to an optionally substitutedhydrocarbon group containing at least one carbon-carbon triple bondbetween the carbon atoms and containing up to 5 carbon atoms joinedtogether. The alkynyl hydrocarbon group may be straight-chained,branched or cyclic. Any substituents are selected from the groupconsisting of halogen, C₁₋₄ alkyl, OCF₃, CF₃, OMe, CN, OSO₂ R and NO₂,wherein R represents C₁₋₄ alkyl or C₃₋₆ cycloalkyl.

The compounds of the present invention may contain one or moreasymmetric carbon atoms and may exist in racemic and optically activeforms. All of these compounds and diastereomers are contemplated to bewithin the scope of the present invention.

Preferred compounds of the present inventions include:

-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;-   N-[(1,1-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;-   N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;    and-   N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Pharmaceutically acceptable salts are non-toxic salts in the amounts andconcentrations at which they are administered.

Pharmaceutically acceptable salts include acid addition salts such asthose containing sulfate, hydrochloride, fumarate, maleate, phosphate,sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfamate and quinate. A preferred salt is a hydrochloride.Pharmaceutically acceptable salts can be obtained from acids such ashydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamicacid, acetic acid, citric acid, lactic acid, tartaric acid, malonicacid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, andquinic acid.

Pharmaceutically acceptable salts also include basic addition salts suchas those containing benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium, ammonium, alkylamine, and zinc, whenacidic functional groups, such as carboxylic acid or phenol are present.

The present invention provides compounds of Formula (I) above which canbe prepared using standard techniques. An overall strategy for preparingpreferred compounds described herein can be carried out as described inthis section. The examples which follow illustrate the synthesis ofspecific compounds. Using the protocols described herein as a model, oneof ordinary skill in the art can readily produce other compounds of thepresent invention.

All reagents and solvents were obtained from commercial vendors.Starting materials were synthesized using standard techniques andprocedures.

A general procedure used to synthesize many of the compounds can becarried out as described in Scheme 1, above: A solution of biarylalcohol in acetone was treated with an appropriate base such as K₂CO₃,heated for 15 min. R-glycidyl nosylate was added and the reactioncontinued overnight to give the corresponding glycidyl ether (Scheme 1).A solution of the substituted glycidyl ether and excess amine (e.g.,1,1-dimethyl-2-(4-methyloxyphenyl)ethylamine) in absolute ethanol,acetonitrile, THF, dioxane or any other similar solvent in the presenceof a suitable catalyst such as LiClO₄ is stirred overnight at reflux.

The product is purified by chromatography. Hydrochloride salts areprepared by treatment of the corresponding free base with HCl either ingas phase or 4M dioxane solution, or any other standard method. Methodsto prepare the biarylphenol are outlined in Schemes 2 and 3.

A 2-fluoro-4-bromobenzonitrile in DMF is treated with boronate, potasiumacetate and catalytic PdCl₂(dppf) at 80° C. After the aryl bromide isconsumed (about 2 h), the reaction is cooled to room temperature andtreated with an aryl acid, 2 M sodium carbonate and additional palladiumcatalysis and then heated for 18 h to give the fluorine substitutedbiaryl product.

The fluorine is displaced with potassium acetate in acetonitrile in thepresence of 18-crown-6 to give the corresponding phenol (see Scheme 2).Alternatively, a 2-methoxy-4-bromobenzonitrile can be coupled to aboronic acid substituted arylcarboxylic acid using a catalytic amounttetrakistriphenylphosphine palladium in a mixture of toluene and 2Msodium carbonate to give a biarylmethyl ether. Deprotection of themethyl ether (e.g., lithium iodide, collidine) followed byesterification gives the corresponding biarylphenol (Scheme 3).

In order to use a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof for the treatment of humans and other mammals,it is normally formulated in accordance with standard pharmaceuticalpractice as a pharmaceutical composition.

The calcilytic compounds can be administered by different routesincluding intravenous, intraperitoneal, subcutaneous, intramuscular,oral, topical (transdermal), or transmucosal administration. Forsystemic administration, oral administration is preferred. For oraladministration, for example, the compounds can be formulated intoconventional oral dosage forms such as capsules, tablets, and liquidpreparations such as syrups, elixirs, and concentrated drops.

Alternatively, injection (parenteral administration) may be used, e.g.,intramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds of the invention are formulated in liquidsolutions, preferably, in physiologically compatible buffers orsolutions, such as saline solution, Hank's solution, or Ringer'ssolution. In addition, the compounds may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms canalso be produced.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration, for example, may be throughnasal sprays, rectal suppositories, or vaginal suppositories.

For topical administration, the compounds of the invention can beformulated into ointments, salves, gels, or creams, as is generallyknown in the art.

The amounts of various calcilytic compounds to be administered can bedetermined by standard procedures taking into account factors such asthe compound IC₅₀, EC₅₀, the biological half-life of the compound, theage, size and weight of the patient, and the disease or disorderassociated with the patient. The importance of these and other factorsto be considered are known to those of ordinary skill in the art.

Amounts administered also depend on the routes of administration and thedegree of oral bioavailability. For example, for compounds with low oralbioavailability, relatively higher doses will have to be administered.

Preferably the composition is in unit dosage form. For oral application,for example, a tablet, or capsule may be administered, for nasalapplication, a metered aerosol dose may be administered, for transdermalapplication, a topical formulation or patch may be administered and fortransmucosal delivery, a buccal patch may be administered. In each case,dosing is such that the patient may administer a single dose.

Each dosage unit for oral administration contains suitably from 0.01 to500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula(I) or a pharmaceutically acceptable salt thereof, calculated as thefree base. The daily dosage for parenteral, nasal, oral inhalation,transmucosal or transdermal routes contains suitably from 0.01 mg to 100mg/Kg, of a compound of Formula(I). A topical formulation containssuitably 0.01 to 5.0% of a compound of Formula (I). The activeingredient may be administered, for example, from 1 to 6 times per day,preferably once, sufficient to exhibit the desired activity, as isreadily apparent to one skilled in the art.

As used herein, “treatment” of a disease includes, but is not limited toprevention, retardation and prophylaxis of the disease.

Diseases and disorders which might be treated or prevented, based uponthe affected cells, include bone and mineral-related diseases ordisorders; hypoparathyroidism; those of the central nervous system suchas seizures, stroke, head trauma, spinal cord injury, hypoxia-inducednerve cell damage, such as occurs in cardiac arrest or neonataldistress, epilepsy, neurodegenerative diseases such as Alzheimer'sdisease, Huntington's disease and Parkinson's disease, dementia, muscletension, depression, anxiety, panic disorder, obsessive-compulsivedisorder, post-traumatic stress disorder, schizophrenia, neurolepticmalignant syndrome, and Tourette's syndrome; diseases involving excesswater reabsorption by the kidney, such as syndrome of inappropriate ADHsecretion (SIADH), cirrhosis, congestive heart failure, and nephrosis;hypertension; preventing and/or decreasing renal toxicity from cationicantibiotics (e.g., aminoglycoside antibiotics); gut motility disorderssuch as diarrhea and spastic colon; GI ulcer diseases; GI diseases withexcessive calcium absorption such as sarcoidosis; autoimmune diseasesand organ transplant rejection; squamous cell carcinoma; andpancreatitis.

In a preferred embodiment of the present invention, the presentcompounds are used to increase serum parathyroid hormone (“PTH”) levels.Increasing serum PTH levels can be helpful in treating diseases such ashypoparathyroidism, osteosarcoma, periodontal disease, fracture,osteoarthritis, rheumatoid arthritis, Paget's disease, humoralhypercalcemia malignancy and osteoporosis.

In a preferred embodiment of the present invention, the presentcompounds are co-administered with an anti-resorptive agent. Such agentsinclude, but are not limited estrogen, 1, 25 (OH)₂ vitamin D3,calcitonin, selective estrogen receptor modulators, vitronectin receptorantagonists, V-H+-ATPase inhibitors, src SH2 antagonists,bisphosphonates and cathepsin K inhibitors.

Another aspect of the present invention describes a method of treating apatient comprising administering to the patient an amount of a presentcompound sufficient to increase the serum PTH level. Preferably, themethod is carried out by administering an amount of the compoundeffective to cause an increase in duration and/or quantity of serum PTHlevel sufficient to have a therapeutic effect.

In various embodiments, the compound administered to a patient causes anincrease in serum PTH having a duration of up to one hour, about one toabout twenty-four hours, about one to about twelve hours, about one toabout six hours, about one to about five hours, about one to about fourhours, about two to about five hours, about two to about four hours, orabout three to about six hours.

In an alternattive embodiment of the present invention, the compoundadministered to a patient causes an increase in serum PTH having aduration of more than about twenty four hours provided that it isco-administered with an anti resorptive agent.

In additional different embodiments, the compound administered to apatient causes an increase in serum PTH of up to two fold, two to fivefold, five to ten fold, and at least 10 fold, greater than peak serumPTH in the patient. The peak serum level is measured with respect to apatient not undergoing treatment.

Composition of Formula (I) and their pharmaceutically acceptable saltswhich are active when given orally can be formulated as syrups, tablets,capsules and lozenges. A syrup formulation will generally consist of asuspension or solution of the compound or salt in a liquid carrier forexample, ethanol, peanut oil, olive oil, glycerine or water with aflavoring or coloring agent. Where the composition is in the form of atablet, any pharmaceutical carrier routinely used for preparing solidformulations may be used. Examples of such carriers include magnesiumstearate, terra alba, talc, gelatin, acacia, stearic acid, starch,lactose and sucrose. Where the composition is in the form of a capsule,any routine encapsulation is suitable, for example using theaforementioned carriers in a hard gelatin capsule shell. Where thecomposition is in the form of a soft gelatin shell capsule anypharmaceutical carrier routinely used for preparing dispersions orsuspensions may be considered, for example aqueous gums, celluloses,silicates or oils, and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of acompound or salt in a sterile aqueous or non-aqueous carrier optionallycontaining a parenterally acceptable oil, for example polyethyleneglycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

Typical compositions for inhalation are in the form of a solution,suspension or emulsion that may be administered as a dry powder or inthe form of an aerosol using a conventional propellant such asdichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent, forexample polymeric glycols, gelatins, cocoa-butter or other low meltingvegetable waxes or fats or their synthetic analogs.

Typical dermal and transdermal formulations comprise a conventionalaqueous or non-aqueous vehicle, for example a cream, ointment, lotion orpaste or are in the form of a medicated plaster, patch or membrane.

Preferably the composition is in unit dosage form, for example a tablet,capsule or metered aerosol dose, so that the patient may administer asingle dose.

No unacceptable toxological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

The biological activity of the compounds of Formula (I) are demonstratedby the following tests:

(I) Calcium Receptor Inhibitor Assay

Calcilytic activity was measured by determining the IC₅₀ of the testcompound for blocking increases of intracellular Ca²⁺ elicited byextracellular Ca²⁺ in HEK 293 4.0-7 cells stably expressing the humancalcium receptor. HEK 293 4.0-7 cells were constructed as described byRogers et al., J. Bone Miner. Res. 10 Suppl. 1:S483, 1995 (herebyincorporated by reference herein). Intracellular Ca²⁺ increases wereelicited by increasing extracellular Ca²⁺ from 1 to 1.75 mM.Intracellular Ca²⁺ was measured using fluo-3, a fluorescent calciumindicator.

The procedure was as follows:

1. Cells were maintained in T-150 flasks in selection media (DMEMsupplemented with 10% fetal bovine serum and 200 ug/mL hygromycin B),under 5% CO₂:95% air at 37° C. and were grown up to 90% confluency.

2. The medium was decanted and the cell monolayer was washed twice withphosphate-buffered saline (PBS) kept at 37° C. After the second wash, 6mL of 0.0₂% EDTA in PBS was added and incubated for 4 minutes at 37° C.Following the incubation, cells were dispersed by gentle agitation.

3. Cells from 2 or 3 flasks were pooled and pelleted (100×g). Thecellular pellet was resuspended in 10-15 mL of SPF-PCB+ and pelletedagain by centrifugation. This washing was done twice.

Sulfate- and phosphate-free parathyroid cell buffer (SPF-PCB) contains20 mM Na-Hepes, pH 7.4, 126 mM NaCl, 5 mM KCl, and 1 mM MgCl₂. SPF-PCBwas made up and stored at 4° C. On the day of use, SPF-PCB wassupplemented with 1 mg/mL of D-glucose and 1 mM CaCl₂ and then splitinto two fractions. To one fraction, bovine serum albumin (BSA; fractionV, ICN) was added at 5 mg/mL (SPF-PCB+). This buffer was used forwashing, loading and maintaining the cells. The BSA-free fraction wasused for diluting the cells in the cuvette for measurements offluorescence.

4. The pellet was resuspended in 10 mL of SPF-PCB+ containing 2.2 uMfluo-3 (Molecular Probes) and incubated at room temperature for 35minutes.

5. Following the incubation period, the cells were pelleted bycentrifugation. The resulting pellet was washed with SPF-PCB+. Afterthis washing, cells were resuspended in SPF-PCB+ at a density of 1-2×106cells/mL.

6. For recording fluorescent signals, 300 uL of cell suspension werediluted in 1.2 mL of SPF buffer containing 1 mM CaCl₂ and 1 mg/mL ofD-glucose. Measurements of fluorescence were performed at 37° C. withconstant stirring using a spectrofluorimeter. Excitation and emissionwavelengths were measured at 485 and 535 nm, respectively. To calibratefluorescence signals, digitonin (5 mg/mL in ethanol) was added to obtainFmax, and the apparent Fmin was determined by adding Tris-EGTA (2.5 MTris-Base, 0.3 M EGTA). The concentration of intracellular calcium wascalculated using the following equation: Intracellularcalcium=(F-F_(min)/F_(max))×K_(d); where K_(d)=400 nM.

7. To determine the potential calcilytic activity of test compounds,cells were incubated with test compound (or vehicle as a control) for 90seconds before increasing the concentration of extracellular Ca²⁺ from 1to 2 mM. Calcilytic compounds were detected by their ability to block,in a concentration-dependent manner, increases in the concentration ofintracellular Ca²⁺ elicited by extracellular Ca²⁺.

In general, those compounds having lower IC₅₀ values in the CalciumReceptor Inhibitor Assay are more preferred compounds. Compounds havingan IC₅₀ greater than 50 uM were considered to be inactive. Preferredcompounds are those having an IC₅₀ Of 10 uM or lower, more preferredcompounds have an IC₅₀ of 1 uM, and most preferred compounds have anIC₅₀ of 0.1 uM or lower.

(II) Calcium Receptor Binding Assay

HEK 293 4.0-7 cells stably transfected with the Human ParathyroidCalcium Receptor (“HuPCaR”) were scaled up in T180 tissue cultureflasks. Plasma membrane is obtained by polytron homogenization or glassdouncing in buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 3 mM MgCl₂) in thepresence of a protease inhibitor cocktail containing 1 uM Leupeptin,0.04 uM Pepstatin, and 1 mM PMSF. Aliquoted membrane was snap frozen andstored at −80° C. ³H labeled compound was radiolabeled to aradiospecific activity of 44 Ci/mmole and was aliquoted and stored inliquid nitrogen for radiochemical stability.

A typical reaction mixture contains 2 nM ³H compound((R,R)-N-4′-Methoxy-t-3-3′-methyl-1′-ethylphenyl-1-(1-naphthyl)ethylamine),or ³H compound(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(4-methoxyphenyl)ethylamine4-10 ug membrane in homogenization buffer containing 0.1% gelatin and10% EtOH in a reaction volume of 0.5 mL. Incubation is performed in12×75 polyethylene tubes in an ice water bath. To each tube 25 uL oftest sample in 100% EtOH is added, followed by 400 uL of cold incubationbuffer, and 25 uL of 40 nM ³H-compound in 100% EtOH for a finalconcentration of 2 nM. The binding reaction is initiated by the additionof 50 uL of 80-200 ug/mL HEK 293 4.0-7 membrane diluted in incubationbuffer, and allowed to incubate at 4° C. for 30 min. Wash buffer is 50mM Tris-HCl containing 0.1 % PEI. Nonspecific binding is determined bythe addition of 100-fold excess of unlabeled homologous ligand, and isgenerally 20% of total binding. The binding reaction is terminated byrapid filtration onto 1 % PEI pretreated GF/C filters using a BrandelHarvestor. Filters are placed in scintillation fluid and radioactivityassessed by liquid scintillation counting.

EXAMPLES

Nuclear magnetic resonance spectra were recorded at either 250 or 400MHz using, respectively, a Bruker AM 250 or Bruker AC 400 spectrometer.CDCl₃ is deuteriochloroform, DMSO-d₆ is hexadeuteriodimethylsulfoxide,and CD₃OD is tetradeuteriomethanol. Chemical shifts are reported inparts per million (●) downfield from the internal standardtetramethylsilane. Abbreviations for NMR data are as follows: s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets,dt=doublet of triplets, app=apparent, br=broad. J indicates the NMRcoupling constant measured in Hertz. Continuous wave infrared (IR)spectra were recorded on a Perkin-Elmer 683 infrared spectrometer, andFourier transform infrared (FTIR) spectra were recorded on a NicoletImpact 400 D infrared spectrometer. IR and FTIR spectra were recorded intransmission mode, and band positions are reported in inversewavenumbers (cm⁻¹). Mass spectra were taken on either VG 70 FE, PE SyxAPI III, or VG ZAB HF instruments, using fast atom bombardment (FAB) orelectrospray (ES) ionization techniques. Elemental analyses wereobtained using a Perkin-Elmer 240C elemental analyzer. Melting pointswere taken on a Thomas-Hoover melting point apparatus and areuncorrected. All temperatures are reported in degrees Celsius.

Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layerplates were used for thin layer chromatography. Both flash and gravitychromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh)silica gel. Analytical and preparative HPLC were carried out on Raininor Beckman chromatographs. ODS refers to an octadecylsilyl derivatizedsilica gel chromatographic support. 5μ Apex-ODS indicates anoctadecylsilyl derivatized silica gel chromatographic support having anominal particle size of 5μ, made by Jones Chromatography, Littleton,Colo. YMC ODS-AQ® is an ODS chromatographic support and is a registeredtrademark of YMC Co. Ltd., Kyoto, Japan. PRP-1® is a polymeric(styrene-divinylbenzene) chromatographic support, and is a registeredtrademark of Hamilton Co., Reno, Nev.) Celite® is a filter aid composedof acid-washed diatomaceous silica, and is a registered trademark ofManville Corp., Denver, Colo. Following the general procedure describedabove the following compounds have been synthesized:

Example 1 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

a) 5-(4-Cyano-3-fluoro-phenyl)-nicotinic acid ethyl ester

A solution of 2-fluoro-4-bromobenzonitrile in DMF is treated withpotassium acetate, bispinacolatoboronate (1.1 equiv.) and catalyticPdCl₂(dppf) and heated for 2 h at 80° C. The reaction mixture is cooledto room temperature and 4-bromonicotinic acid (1 equiv.) is added alongwith fresh catalyst and 2 M Na₂CO₃ and the resulting mixture is stirredat 80° C. for 18 h. Solvent is removed and the residue is treated with4N HCl/dioxane in refluxing ethanol for 18 h. The reaction is evaporatedand the residue in ethyl acetate is washed with NaHCO₃ (aqueous), driedover MgSO₄ and evaporated to give 5-(4-cyano-3-fluoro-phenyl)-nicotinicacid ethyl ester.

b) 5-(4-Cyano-3-hydroxy-phenyl)-nicotinic acid ethyl ester

A mixture of 5-(4-cyano-3-fluoro-phenyl)-nicotinic acid ethyl ester fromExample 1a, potassium acetate (2 equiv.), and 18-crown-6 ether (2equiv.) in MeCN is heated at reflux in 36 h. The mixture is cooled,aqueous sodium carbonate is added and stirred at room temperatureovernight. The mixture was extracted with ether (discarded). The aqueouslayer is neutralized with 1N HCl, extracted with EtOAc, dried overMgSO₄, and concentrated. Purification by flash column chromatographygives 5-(4-cyano-3-hydroxy-phenyl)-nicotinic acid ethyl ester.

c) 5-(4-Cyano-3-R-oxiranylmethoxy-phenyl)-nicotinic acid ethyl ester

A mixture of the 5-(4-cyano-3-hydroxy-phenyl)-nicotinic acid ethyl esterfrom Example 1b (1 equiv.), potassium carbonate (2 equiv.), andR-glycidyl-3-nitrobenzenesulfonate (1 equiv.) in acetone is heated atreflux in 24 h. The mixture is cooled, concentrated, taken up in H₂O andis extracted with ethyl acetate. The organic extracts are washed withbrine, dried over MgSO₄, concentrated to afford5-(4-cyano-3-R-oxiranylmethoxy-phenyl)-nicotinic acid ethyl ester.

d)N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]1,1-dimethyl-2-(5-chlorothienyl)ethylamine

A mixture of 5-(4-cyano-3-R-oxiranylmethoxy-phenyl)-nicotinic acid ethylester from Example 1c (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-2-(5-chlorothienyl)ethylamine (1.1 equiv.) in dioxane isheated at reflux for 48 h. The mixture is cooled, concentrated, taken upin H₂O, extracted with CH₂Cl₂. The organic extracts are washed withbrine, dried over MgSO₄, concentrated, and purified by flash columnchromatography to affordN-[(2R)-hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine.

Example 2 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

To a stirred solution of the compound from Example 1d in dioxane isadded 2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamineas its bis-hydrochloride salt.

Example 3 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine

A mixture of the compound from Example 1c (1 equiv.), lithiumperchlorate (1 equiv.), and 1,1-dimethyl-2-(indan-2-yl)ethylamine (1.1equiv.) in dioxane is heated at reflux for 48 h. The mixture is cooled,concentrated, taken up in H₂O, extracted with CH₂Cl₂. The organicextracts are washed with brine, dried over MgSO₄, concentrated, andpurified by flash column chromatography toN-[(2R)-hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine.

Example 4 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine

To a stirred solution of the compound from Example 3 in dioxane is added2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine.

Example 5 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine

A mixture of Example 1c (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-4-(methoxyphenyl)ethylamine (1.1 equiv.) in dioxane isheated at reflux for 48 h. The mixture is cooled, concentrated, taken upin H₂O, extracted with CH₂Cl₂. The organic extracts are washed withbrine, dried over MgSO₄, concentrated, purified by flash columnchromatography to affordN-[(2R)-hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Example 6 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine

To a stirred solution of the compound from Example 5 in dioxane is added2.5N NaOH (aqueous), and is stirred at room temperature for 18 h. Themixture is concentrated, taken up in H₂O, acidified with 2N HCl to pH=4to giveN-[(2R)-hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Example 7 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

a) 6-(4-Cyano-3-R-oxiranylmethoxy-phenyl)-pyridine-2-carboxylic acidethyl ester.

Utilizing the procedure outlined in Example 1a-c but replacing4-bromonicotinic acid with 6-bromopicolinic acid in Example 1a give6-(4-Cyano-3-R-oxiranylmethoxy-phenyl)-pyridine-2-carboxylic acid ethylester.

b)N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

A mixture of Example 7b (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-2-(5-chlorothienyl)ethylamine (1.1 equiv.) in dioxane isheated at reflux for 48 h. The mixture is cooled, concentrated, taken upin H₂O, extracted with CH₂Cl₂. The organic extracts are washed withbrine, dried over MgSO₄, concentrated, and purified by flash columnchromatography to affordN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine.

Example 8 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

To a stirred solution of the compound from Example 7b in dioxane isadded 2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamineas its bis-hydrochloride salt.

Example 9 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine

A mixture of Example 7b (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-2-(indan-2-yl)ethylamine (1.1 equiv.) in dioxane is heatedat reflux for 48 h. The mixture is cooled, concentrated, taken up inH₂O, extracted with CH₂Cl₂. The organic extracts are washed with brine,dried over MgSO₄, concentrated, and purified by flash columnchromatography to affordN-[(2R)-hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine.

Example 10 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine

To a stirred solution of the compound from Example 9 in dioxane is added2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine.

Example 11 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine

A mixture of Example 7b (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-4-(methoxyphenyl)ethylamine (1.1 equiv.) in dioxane isheated at reflux for 48 h. The mixture is cooled, concentrated, taken upin H₂O, extracted with CH₂Cl₂. The organic extracts are washed withbrine, dried over MgSO₄, concentrated, and purified by flash columnchromatography to affordN-[(2R)-hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Example 12 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine

To a stirred solution of the compound from Example 11 in dioxane isadded 2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Example 13 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

a) 2-Bromo-3-methoxy-pyridine

A solution of 2-bromo-3-hydroxy-pyridine (1 equiv., Aldrich ChemicalCompany) in THF is treated with NaH (1 equiv.) at 0° C. for 30 min.Methyl iodide (1 equiv.) is added and stirred for 18 h. The reactionmixture is evaporated, the residue is taken into ethyl acetate, washedwith 5% Na₂CO₃ (aqueous), dried over MgSO₄ and evaporated to give2-bromo-3-methoxy-pyridine.

b) 3-Methoxy-pyridine-2-carbonitrile

A solution of 2-bromo-3-methoxy-pyridine from Example 13a in DMSO istreated with NaCN at 120° C. for 18 h. The reaction mixture isevaporated, the residue is taken into ethyl acetate, washed with 5%Na₂CO₃ (aqueous), dried over MgSO₄ and evaporated to give3-methoxy-pyridine-2-carbonitrile.

c) 6-Bromo-3-methoxy-pyridine-2-carbonitrile

A solution of 3-methoxy-pyridine-2-carbonitrile from Example 13b in CCl₄is treated with N-bromosuccinimide (1 equiv.) and catalytic2,2-azobisisobutyronitrile and is heated at reflux for 18 h. Thereaction mixture is evaporated, the residue is taken into ethyl acetate,washed with 5% Na₂CO₃ (aqueous), 5 % Na₂S₂O₃ (aqueous), dried over MgSO₄and evaporated to give 6-bromo-3-methoxy-pyridine-2-carbonitrile.

d) 4-(6-Cyano-5-methoxy-pyridin-2-yl)-benzoic acid ethyl ester

A solution of 6-bromo-3-methoxy-pyridine-2-carbonitrile from Example 13cin toluene is treated with 2 M Na₂CO₃ (aqueous), 4-carboxylphenylboronicacid (1 equiv.), ethanol and catalytic (Ph₃P)₄Pd) and is heated at 80°C. for 18 h. The reaction mixture is evaporated and the residue isdissolved in ethanol with 4 N HCl in dioxane and heated at reflux for 18h. The reaction mixture is evaporated, the residue taken into ethylacetate, washed with 5% Na₂CO₃ (aqueous), dried over MgSO₄ andevaporated to give 4-(6-cyano-5-methoxy-pyridin-2-yl)-benzoic acid ethylester.

e) 4-(6-Cyano-5-hydroxy-pyridin-2-yl)-benzoic acid ethyl ester

A solution of 4-(6-cyano-5-methoxy-pyridin-2-yl)-benzoic acid ethylester from Example 13d in collidine is treated with LiI and heated at120° C. for 24 h. The reaction mixture is evaporated, the residue istaken into water and neutralized with 1 N HCl. The resulting precipitateis collected and dried to give4-(6-cyano-5-hydroxy-pyridin-2-yl)-benzoic acid ethyl ester.

f) 4-(6-Cyano-5-oxiranylmethoxy-pyridin-2-yl)-benzoic acid ethyl ester

A mixture of the compound from Example 13e (1 equiv.), potassiumcarbonate (2 equiv.), and R-glycidyl-3-nitrobenzenesulfonate (1 equiv.)in acetone is heated at reflux in 24 h. The mixture was cooled,concentrated, is taken up in H₂O and is extracted with ethyl acetate.The organic extracts are washed with brine, dried over MgSO₄, andconcentrated to afford4-(6-cyano-5-oxiranylmethoxy-pyridin-2-yl)-benzoic acid ethyl ester.

g)N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

A mixture of Example 13f (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-2-(5-chlorothienyl)ethylamine (1.1 equiv.) in dioxane isheated at reflux in 48 h. The mixture is cooled, concentrated, taken upin H₂O, extracted with CH₂Cl₂. The organic extracts are washed withbrine, dried over MgSO₄, concentrated and purified by flash columnchromatography to affordN-[(2R)-hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine.

Example 14 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine

To a stirred solution of the compound from Example 13g in dioxane isadded 2.5N NaOH (aqueous), and is stirred at room temperature for 18 h.The mixture is concentrated, taken up in H₂O, acidified with 2N HCl topH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine.

Example 15 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine

A mixture of Example 13e (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-2-(indan-2-yl)ethylamine (1.1 equiv.) in dioxane is heatedat reflux for 48 h. The mixture is cooled, concentrated, taken up inH₂O, extracted with CH₂Cl₂. The organic extracts are washed with brine,dried over MgSO₄, concentrated, purified by flash column chromatographyto affordN-[(2R)-hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine,.

Example 16 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]]-1,1-dimethyl-2-(indan-2-yl)ethylamine

To a stirred solution of the compound from Example 15 in dioxane isadded 2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]]-1,1-dimethyl-2-(indan-2-yl)ethylamine.

Example 17 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine

A mixture of Example 13e (1 equiv.), lithium perchlorate (1 equiv.), and1,1-dimethyl-4-(methoxyphenyl)ethylamine (1.1 equiv.) in dioxane isheated at reflux for 48 h. The mixture is cooled, concentrated, taken upin H₂O, extracted with CH₂Cl₂. The organic extracts are washed withbrine, dried over MgSO₄, concentrated, purified by flash columnchromatography to affordN-[(2R)-hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Example 18 Preparation ofN-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine

To a stirred solution of the compound from Example 5 in dioxane is added2.5N NaOH (aqueous). The resulting solution is stirred at roomtemperature for 18 h. The mixture is concentrated, taken up in H₂O,acidified with 2N HCl to pH=4 to giveN-[(2R)-hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.

Example 19

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of Formula (I) inpolyethylene glycol with heating. This solution is then diluted withwater for injections (to 100 ml). The solution is then rendered sterileby filtration through a 0.22 micron membrane filter and sealed insterile containers.

All publications, including but not limited to patents and patentapplications cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference as though fullyset forth.

1. A compound selected from the group consisting of:N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-ethylcarboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[5-carboxyl]-3-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-ethylcarboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-5-[[3-carboxyl]-2-pyridyl]phenoxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxy]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(5-chlorothienyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]]-1,1-dimethyl-2-(indan-2-yl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-ethylcarboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine;N-[(2R)-Hydroxy-3-[[2-cyano-4-[[4-carboxyl]phenyl]-3-pyridyloxy]propyl]]-1,1-dimethyl-4-(methoxyphenyl)ethylamine.2. A method of treating osteoporosis in a mammal comprisingadministering therapeutically effective amount of compound according toclaim
 1. 3. A method according to claim 2 wherein the compound isco-administered with an anti-resorptive agent.
 4. A method according toclaim 3 wherein the anti-resorptive agent is selected from the groupconsisting of estrogen, 1, 25 (OH)₂ vitamin D3, calcitonin, selectiveestrogen receptor modulators, vitronectin receptor antagonists,V-H+-ATPase inhibitors, src SH2 antagonists, bisphosphonates andcathepsin K inhibitors.